Formation Mechanism and Performance of Films through Reduced Glutathione-Induced Lysozyme Interface Assembly

In this study, the mechanism of film formation through glutathione-induced lysozyme interface assembly was verified by spectroscopic methods, and the formed film was characterized by ellipsometry. N-(1-pyrenyl)maleimide (NPM) staining demonstrated that reduced glutathione broken the disulfide bonds of lysozyme leading to release of free sulfhydryl groups. Together, tryptophan fluorescence spectroscopy and lysozyme activity assay verified that the disulfide bond Cys6-Cys127 in lysozyme was broken. In addition, the unfolded lysozyme was found to still have antibacterial activity. The lysozyme film was transparent and colorless. Atom force microscopy (AFM) and transmission electron microscopy (TEM) revealed that it was composed of lysozyme aggregates with a uniform, smooth and dense surface. The results of X-ray photoelectron spectroscopy (XPS) and laser confocal Raman spectroscopy (LCRS) indicated that the lysozyme nanofilm might possess interfacial adhesion. The results of ellipsometry showed that the maximum thickness of the film was approximately 900 nm, and did not increase without limit with the increase in incubation time or the concentration of the reactants. Its stiffness value, elastic modulus and hardness were 29.32–34.21 μN/nm, 24.00–27.93 GPa, and 0.44–0.49 GPa, respectively, which were in the strength range of the protein film. The three-phase contact angle of the film was around 70°, indicating good hydrophilicity. Its thermal stability was high, and no degradation occurred at 200 ℃. The preparation of the lysozyme nanofilm provides a good idea and reference for the construction of novel edible coatings.